Precision to the very end: what happens when two replication forks converge during termination?

DNA encodes the information that provides the basis for all life. For cell division to take place the entire DNA of a cell has to be fully duplicated, ensuring that both the mother and the daughter cell can get one complete copy. In addition, the copy made has to be identical to the original. Any changes to the DNA can potentially be harmful, as they will alter how cells function, or even lead to cell death. On a single cell level, accuracy of the duplication process is normally so high that not a single error is made when the millions of DNA base pairs are copied. This extraordinary high level of accuracy is achieved by a network of different processes that regulate the duplication process and choreograph segregation of the two complete copies into mother and daughter. However, mutations in the DNA can inactivate this network of processes and it is a hallmark of cancer cells that they grow in an uncontrolled way.

We are studying the final stages of the duplication process. Replication of DNA is initiated at specific sites called origins of replication. Two complex machines termed replication forks are recruited to these origins. These replication forks are capable of copying the DNA with high precision. While doing so they move in opposite direction at very high speed until they meet another replication fork coming the opposite way. Our research has revealed that the collision of two fast moving replication forks has the potential to corrupt the DNA and introduce mutations. This cause of mutation is unexpected, because the fusion of two such replication forks is a fundamental process when DNA is copied, and we have identified a number of pathways that can prevent the harmful consequences of fork fusions.

Currently we are using the bacterium Escherichia coli to gain a better insight into the mechanics of such fork fusion events. In E. coli only one such fusion event occurs, as the entire DNA is copied by only two replication forks. In our own cells the duplication process is initiated at hundreds of origins, leading to hundreds of fusion events, making studies much more complex. The relative simplicity of fork fusions in E. coli will allow us to study these events at very high detail with many different tools. For example, we will use recently developed microscopy techniques which allow us to directly visualise and track single replication forks inside living cells to study what happens if forks fuse and to identify how these fusion events can cause corruption of the DNA. Our results will allow us to generate a model of how the duplication process of DNA is normally brought to an orderly completion, which will help us to understand this process in our own cells, and our work will reveal whether it might contribute to the development of cancer and ageing.

All organisms need to replicate their chromosomes with high fidelity to ensure that the genetic information passed on to the next generation is sufficiently accurate. Chromosome duplication initiates at defined origins, with two replication forks proceeding in opposite directions. DNA replication terminates when a replication fork meets the end of a chromosome or another fork travelling in the opposite direction. We have demonstrated in Escherichia coli that fork fusion events, if not processed correctly, result in surprisingly severe consequences, such as persistent over-replication of the chromosome, increased recombination and chromosome segregation defects. Thus, for the accurate completion of genome duplication the fusion of two converging forks must be carefully controlled, a theme also emerging for the hundreds of fork fusion events in eukaryotic cells.

While we have identified some of the pathologies that arise if fork fusions are not processed correctly, our understanding of the molecular mechanics of fork fusion is still limited. Here we propose to use a combined in vivo and in vitro approach in E. coli to directly analyse the protein dynamics and the DNA intermediates arising at fusing forks. We will investigate how fork fusion intermediates are processed and what happens when this processing goes awry, and we will determine how termination is choreographed in the context of whole chromosome dynamics, segregation and cell division. These analyses will provide a detailed view of replication termination and how the incorrect processing of fork fusions can result in pathologies. Our data will form an important foundation for the understanding of how the hundreds of fork fusions in eukaryotic cells are achieved and how their processing contributes towards maintaining genomic stability. Insight into the factors maintaining genomic integrity is much needed for our understanding of cancer, ageing and many hereditary diseases.

The described programme will provide fundamental insights into what happens when two complex and fast moving replication forks converge and finally fuse. The fusion of replication forks is a necessity of DNA replication and therefore a fundamental aspect of the cell cycle in all organisms. In addition, the implications of our research address fundamental questions of the evolution of chromosomal architecture and replication speed in pro- and eukaryotes. Our studies will shed light on the mechanisms that have evolved to deal with the intermediates arising as forks fuse to allow duplication of the entire chromosome with a fidelity sufficient to avoid significant corruption of the genomic information.

Our recent research in E. coli has demonstrated that fork fusions can result in pathological consequences such as extensive over-replication of the chromosome, increased recombination and problems with cell cycle progression. It will be important to establish a mechanistic basis of how fork fusions are processed to limit genomic instability, as mistakes made during DNA replication are crucial in the development of genetic disease and other mutation-driven problems such as cancer. Clinicians and scientists with interests in hereditary diseases will therefore benefit from our fundamental studies. The general mechanics of DNA duplication is similar in all living organisms and studies in bacterial model organisms have provided many paradigms for understanding these processes in more complex systems. Currently, little research is carried out on replication fork fusions and the potential impact on genomic stability and our results will significantly contribute towards strengthening the international competitiveness of the research on DNA replication and genomic stability carried out within the UK.

Our studies will also have impact on medical and biotechnological applications. Streptomycetes are an important sources for antibiotics. Their chromosome is normally linear, in contrast to many other bacterial species, but it can circularise. It was noted before that this circularisation results in a significant increase of chromosomal instability and it is very tempting to speculate that this instability is a consequence of aberrantly processed fork fusion intermediates. Our work therefore has the potential to be of relevance for technical applications such as large scale culturing of Streptomycetes for production of antibiotics or other secondary metabolites of biological or chemical relevance. Furthermore, we have identified RecG helicase as one of the key players in defusing potentially harmful fork fusion intermediates. The combined deletion of recG and other genes involved in processing fork fusion intermediates is lethal in E. coli. RecG, while being present in most bacterial species, has no known counterpart in mammalian cells. Thus, the proposed work may be of long-term benefit to pharmaceutical applications aiming to develop new targets for inhibition of pathogenic bacteria. Thus, our studies will have relevance to medicine, agriculture and industry.

The proposed research will combine complex biochemical work, molecular genetics and cell biology studies as well as computer modelling approaches to whole genome replication, resulting in significant cross-disciplinary training of all scientists involved. This will strengthen the scientifically-literate workforce and therefore the international competitiveness of the UK. Understanding how healthy organisms maintain genomic stability and cell division, and what happens when these processes go awry, will have long-term benefits to the health and well-being of the UK population. In addition, all researchers of this project will be well-placed to engage with the public to communicate the links between genomes, mutation and the genetic basis of disease, topics of general interest to the public.